Important anti-tumor agents exist known as dimeric catharanthus alkaloids also known as the vinca alkaloids. The vinca alkaloids are obtained from the fractionation of extracts of the Periwinkle Plant (Vinca rosea) which is a species of myrtle. There are four generally known extracts that are active dimeric alkaloids which include vinblastine, vincristine, vinleurosine and vinrosidine. Vinblastine and vincristine are the two most common and important clinical agents. Vinblastine is primarily used in combination with bleomycin and cisplatin in the treatment of metastatic testicular tumors. Vinblastine also promotes beneficial responses in lymphoma and is active in Kaposi's sarcoma, neuroblastoma, and letterer-siwe disease (Histiocytosis X). It is also known to be active in carcinoma of the breast and choriocarcinoma in women.
Vincristine is the preferred treatment in childhood leukemia. Vincristine also has similar clinical activity to that of vinblastine. For example vincristine is effective in the treatment of Hodgkin's Disease. It is also useful in non-Hodgkin's lymphoma when used in combination with such agents as bleomycin and doxorubicin. Vincristine is also used to elicit responses in patients with neoplasms such as Wilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, carcinoma of the breast, bladder and tumors that present themselves in male and female reproductive systems. Vincristine is also used in combination with doxorubicin and bleomycin in the treatment of Kaposi's sarcoma. The effectiveness of the vinca-alkaloids is in their ability to cause mitotic arrest at metaphase in dividing cells during the cell cycle, thus arresting cell division in metaphase. Although the above agents have been effective as anti-cancer agents there have been associated with the use of these agents serious cytotoxic effects on normal cells. For example, bone-marrow depression is common. Neurotoxicity is another serious side effect associated with Vincristine. Alopecia and hair loss are also associated with the use of vinblastine and vincristine. Other toxicities include anemia, polyuria, dysuria, gastrointestinal irregularities, thrombocytopenia and cardiotoxicity.
In an effort to decrease the toxicity of the vinca-alkaloids and to increase their therapeutic effectiveness, workers have attempted to utilize liposomal formulations of vincristine and vinblastine to overcome these problems.
Liposomes are microscopic vesicles made, in part, from phospholipids which form closed, fluid filled spheres when dispersed with water. Phospholipid molecules are polar, having a hydrophilic ionizable head group and two hydrophobic tails consisting of long fatty acid chains. Thus, when sufficient phospholipid molecules are present with water, the tails spontaneously associate to exclude water while the hydrophilic phosphate heads interact with water. The result is a bilayer membrane in which the fatty acid tails converge in the newly formed membrane's interior and the polar heads point in opposite directions toward an aqueous medium. These bilayer membranes can be caused to form closed spheres known as liposomes. The polar heads at the inner surface of the membrane point toward the aqueous interior of the liposome. At the opposite surface of the spherical membrane, the polar heads interact with the surrounding aqueous medium. As the liposomes are formed, water soluble molecules can be incorporated into the aqueous interior, and lipophilic molecules will tend to be incorporated into the lipid bilayer. Liposomes may be either multilamellar, like an onion with liquid separating many lipid bilayers, or unilamellar, with a single bilayer surrounding an entirely aqueous liquid center.
There are many types of liposome preparation techniques which may be employed and which produce various types of liposomes. These can be selected depending on the use, the chemical intended to be entrapped, and the type of lipids used to form the bilayer membrane. The requirements which must be considered in producing a liposome preparation are similar to those of other controlled release mechanisms. They are as follows: (1) high percent of chemical entrapment; (2) increased chemical stability; (3) low chemical toxicity; (4) rapid method of production; and (5) reproducible size distribution.
The first method described to encapsulate chemicals in liposomes involved production of multilamellar vesicles (MLVs). Methods for encapsulating chemicals in MLVs are known in the art.
Liposomes can also be formed as unilamellar vesicles (UVs), which generally have a size less than 1 .mu.m. There are several techniques known in the art which are used to produce unilamellar liposomes.
Smaller unilamellar vesicles can be formed using a variety of techniques. By dissolving phospholipids in ethanol and injecting them into a buffer, the lipids will spontaneously rearrange into unilamellar vesicles. This provides a simple method to produce UVs which have internal volumes similar to that of those produced by sonication (0.2-0.5 L/mol/lipid). Sonication, extrusion (through filters) of MLVs also results in dispersions of UVs having diameters of up to 0.2 .mu.m, which appear as clear or translucent suspensions.
Another common method for producing small UVs is the detergent removal technique. Phospholipids are solubilized in either ionic or non-ionic detergents such as cholates, Triton X, or n-alkylglucosides. The drug is then mixed with the solubilized lipid-detergent micelles. Detergent is then removed by one of several techniques: dialysis, gel filtration, affinity chromatography, centrifugation, ultrafiltration. The size distribution and entrapment efficiencies of the UVs produced this way will vary depending on the details of the technique used.
Smaller unilamellar vesicles can be formed using a variety of techniques, such as applying a force sufficient to reduce the size of the liposomes and or produce smaller unilamellar vesicles. Such force can be produced by a variety of methods, including homogenization, sonication or extrusion (through filters) of MLV's. These methods result in dispersions of UVs having diameters of up to 0.2 .mu.m, which appear as clear or translucent suspensions. Other standard methods for the formation of liposomes are know in the art, for example, methods for the commercial production of liposomes include the homogenization procedure described in U.S. Pat. No. 4,753,788 to Gamble, a preferred technique, and the method described in U.S. Pat. No. 4,935,171 to Bracken, which are incorporated herein by reference.
The therapeutic use of liposomes includes the delivery of drugs which are normally toxic in the free form. In the liposomal form the toxic drug may be directed away from the sensitive tissue and targeted to selected areas. Liposomes can also be used therapeutically to release drugs, over a prolonged period of time, reducing the frequency of administration. In addition, liposomes can provide a method for forming an aqueous dispersion of hydrophobic drugs for intravenous delivery.
When liposomes are used to target encapsulated drugs to selected host tissues, and away from sensitive tissues, several techniques can be employed. These procedures involve manipulating the size of the liposomes, their net surface charge as well as the route of administration. More specific manipulations have included labeling the liposomes with receptors or antibodies for particular sites in the body. The route of delivery of liposomes can also affect their distribution in the body. Passive delivery of liposomes involves the use of various routes of administration, e.g., intravenous, subcutaneous and topical. Each route produces differences in localization of the liposomes. Two common methods used to actively direct the liposomes to selected target areas are binding either antibodies or specific receptor ligands to the surface of the liposomes. Antibodies are known to have a high specificity for their corresponding antigen and have been shown to be capable of being bound to the surface of liposomes, thus increasing the target specificity of the liposome encapsulated drug.
Since the chemical composition of many drugs precludes their intravenous administration, liposomes can be very useful in adapting these drugs for intravenous delivery. Many hydrophobic drugs, fall into this category because they cannot be easily dissolved in a water-based medium and must be dissolved in alcohols or surfactants which have been shown to cause toxic reactions in vivo. Liposomes, composed of lipids, with or without cholesterol, are nontoxic. Furthermore, since liposomes are made up of amphipathic molecules, they can entrap hydrophilic drugs in their interior space and hydrophobic molecules in their lipid bilayer. Although methods for making liposomes are well known in the art, it is not always possible to determine a working formulation without undue experimentation.
Efforts have been made to increase the therapeutic ability of anticancer or antineoplastic drugs in general and vincristine in particular. Efforts have also been made to reduce the toxic side effects associated with the use of these agents. Further, work has been done to increase the circulation time and accumulation in tumors of the various agents encapsulated within the liposomes. Examples of the work that has been done with tumor uptake is discussed in U.S. Pat. No. 5,019,3692 and U.S. application Ser. No. 07/835931 filed on Jul. 3, 1991 7/3/91 (PCT); both of these documents are incorporated by reference. Specific work performed on liposomal vincristine is also shown in references such as D. Layton and A. Trouet, Europ. J. Cancer (1980) 16:945-50; J. Vaage et al., Int. J. Cancer (1993) 54:/959-64; and L. D. Mayer et al., Cancer Chemother. Phamacol. (1993) 33:/7-24. Much work remains to be done in developing liposomal formulations which are stable on storage and which are capable of enhanced tumor targeting.
Thus, it is a desideratum to provide for a liposomal formulation with enhanced tumor targeting properties and which provides an increase in therapeutic value over free drug and offers decreased toxicity. It is an object of the invention to provide a liposomal formulation that is stable in that the formulation will not aggregate over time.